Patterns of behavior and processes of learning and memory are probably, in large part, based on regulation of neurotransmitter release from nerve terminals. In addition, the cellular etiology of most neurological diseases involves, at some point, inappropriate levels of transmitter release. Thus, beneficial manipulation of behavior memory, learning, and neuropathologies would be enhanced by understanding the molecular mechanisms of the regulation of transmitter release. In many neuronal systems, including the neuromuscular junction and the marine ray electric organ, adenosine inhibits release of transmitter through activation of specific extracellulr presynaptic receptors. The proposed investigations will use immunopurified nerve terminals of the electric organ to study the molecular basis of this regulatory process. Isolated electric organ nerve terminals are well-suited for biochemical scrutiny because they are easily available in quantity, are pure and homogeneous, and are homologous to neuromuscular junction terminals. In addition, complementary electrophysiological studies of release in the intact organ can be performed. Modulation of release by presynaptic receptors is uniquitous in the nervous system and in many cases may proceed through a common mechanism. Briefly, activation of these receptors alters intraterminal c-AMP levels, which in turn mediate the phosphorylation of protein(s) associated with the transmitter release system, thus affecting release. While often hypothesized, this sequence of events has never been fully demonstrated in any vertebrate neuron. A major goal of these proposed studies is to show that adenosine inhibition operates through this mechanism. The recent finding that adenosine increases intraterminal c-AMP motivates the search for which protein(s) is(are) phosphorylated by c-AMP-dependent kinase. Manipulations which circumvent the normal release process and localization of the phosphorylated protein(s) in the nerve terminal by subcellular fractionation will help identify the point in the release system where inhibitory phosphorylation impinges. The phosphorylated component(s) associated with the release system, which may be the Ca++ channel, a Ca++ dependent kinase, the protein synapsin I, or some other unknown protein, will be isolated and characterized. Reconstitution of a simple, functional system containing this protein will initiate investigations to characterize the molecular basis of adenosine-dependent inhibition of its function. Corollary electrophysiological studies of these phenomena in the neuromuscular will be continued.